Project Summary Approximately 12 million people inject drugs globally; 13% of whom are people living with HIV (PLWH). Opioid misuse is a route of HIV acquisition and a barrier to effective antiretroviral therapy (ART). However, it is unclear whether opioid misuse changes the course of HIV pathogenesis and latency. Latency is a barrier to curing HIV because it results in a reservoir of infected quiescent cells that evade the antiviral immune response, are not targeted by ART, and allow HIV viremia to rebound upon treatment interruption. Latent cells are rare and lack identifying biomarkers, making it challenging to quantify and characterize the latent cell reservoir. Our central hypothesis is that opioid misuse exacerbates HIV pathogenesis and the establishment of HIV latency by dysregulating the host immune response. Our overall objective is to exploit transcriptomic information from patient samples to characterize the effects of opioid use disorder on host immune function, HIV replication, and HIV latency. We will leverage four existing NIDA- and NIAAA-funded studies in St. Petersburg, Russia whose participants have well-characterized opioid use. In the R61 phase, we will characterize single cell gene expression and identify dysregulated gene regulatory networks in immune cells associated with opioid misuse in PLWH. We will also perform computational analysis to identify immune cell gene regulatory networks altered by opioid misuse (Aim 1). Next, we will combine single cell RNA-seq and fluorescent in situ hybridization flow cytometry (flow-FISH) to characterize the transcriptome in cell models of latent HIV infection (Aim 2). In the R33 phase, we will use tools developed and optimized in Aim 2 to characterize the latent reservoir in PLWH with and without opioid misuse. We will also validate whether immune cell gene networks altered by opioid misuse influence HIV transcription and replication (Aim 3). Lastly, we will use predictions from computational analysis in Aim 1 to test candidate molecules for their ability to influence HIV transcription and latency among PLWH with and without opioid use. Successful completion of these aims will have significant research and clinical impact by: 1) elucidating how opioid misuse alters HIV pathogenesis; 2) producing novel tools for characterizing HIV latency; and 3) discovering candidate molecules to regulate HIV expression in the context of opioid misuse.